Quantitative laboratory tests with high accuracy and precision are mandatory for the correct diagnosis of coagulation disorders. A blood specimen must be collected aseptically using a disposable plastic syringe, avoiding hemolysis and contamination from the tissue juice. For a special clotting test, the two-syringe technique is recommended.
The internationally accepted procedures (ICSH, ICTH, etc.) recommend an anticoagulant concentration of 3.2% tri-sodium citrate·2H₂O (0.109M). Commercially available reagents for blood coagulation tests are of the two types: activated partial thromboplastin time (APTT) reagent and prothrombin time (PT) reagent. They differ in the source organs from which they are extracted and the purification procedures by which they are prepared. Also, various automated instruments based on several mechanism such as electrical, optical, and mechanical, are commonly used. Although low CVs (coefficient of variation) are obtained when APTT or PT is determined by the use of an automated instrument, the quantitative parameter, such as clotting time and the shape of standard curve, critically depends on the type of reagent or instrument, as well as on the combination of them. Identical end point is not always realized when another reagent or instrument is used for the assay. In this respect standardization of the assay procedures for universal acceptance is storongly desired.
In an immunological method an optimal combination of antigen and antibody concentrations is a prerequisite for a reliable antigen-antibody reaction. In a reaction with a synthetic chromogenic substrate the most crucial factor is the precision in the reaction time. These assays are specific only when cross-reactions of these reagents with other factors present in the specimen is negligible. Because these assays are based on one of the following activities, clotting, immunological, and amidolytic, which may or may not correlated very well with each other, through understanding of the assay specificity is essential for the correct diagnosis.

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In recent papers it is reported that C1INA not only inhibits C1S and C1R, but also inhibits coagulation factors such as XIIa, XIa, kallikrein and plasmin, and Nagaki, et al. in 1977 described the fact that heparin promorted remarkably the inhibitory action of C1INA on C1S. Therefore, it is attempted in this paper to observe the effect of heparin on the inhibitory action of C1INA to coagulation factors, especially to plasma kallikrein, and the following results are obtained;
1) Normal human plasma shows one peak pattern in cross immunoelectrophoresis (CIEP) by the use of anti-C1INA serum. On the contrally, when heparin is added to normal plasma, three peaks appear in electrophoretic pattern, and this finding may indicate the complex formation of C1INA and heparin. If protamin sulfate is added to the mixture of plasma and heparin, the pattern of CIEP returns to one peak figure of normal plasma alone. These findings also suggest that the complex formation of C1INA and heparin is reversible.
2) The inhibitory action of heparin-C1INA complex against human plasma kallikrein in normal plasma and modified plasma including C1INA free plasma, AT III free plasma and both C1INA and AT III free plasma is studied by the use of chromogenic substrate (C-PK). In the cases of normal and AT III free plasma with heparin, the activity of plasma kallikrein reduces rapidly in proportion to the increase of heparin concentration, but in the cases of C1INA free plasma and C1INA-AT III free plasma there are no marked decrease of kallikrein activity in any concentration of heparin.
3) The addition of purified C1INA onto normal plasma decreases slightly the kallikrein activity, but the addition of heparin on the mixture above mentioned increases antikallikrein action of C1INA with enhancement of reaction rate.
4) The influences of heparin added to the mixture of purified human C1INA and plasma kallikrein are observed by chromogenic substrate technique and obtained the result, that heparin accelerates the formation of kallikrein-C1INA complex.
5) It is strongly suggested by the results mentioned above that the function of heparin in so-called heparin therapy for hypercoagulable state including DIC and hereditary angioneurotic edema may related with the accelerating antikallikrein action by heparin-C1INA complex formation.

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Fibrinolysis, coagulation, and kinin system relate to pathogenesis of gastric ulcer. Serum kinin has complicated backgrounds and its changes are not in accordance with pathogenesis of gastric ulcer. The relationships between tissue-fibrinolysis and tissue-kallikrein were studied in tissue-plasminogen activator and tissue-kallikrein.
Recently, kallikrein has been found in stomach tissue. Synthetic substrate, Prolyl-Phenyl-Arginyl-Naphtyl-Ester, was used to determine its activity. It was studied whether its value reflects clearly tissue-kallikrein or not.
Satisfactory results were obtained in the basic study. Then an experiment of stress ulcer due to water immersion was performed with DONRYU rat. In the study on a relationship between tissue-plasminogen activator and tissue-kallikrein during the term from preloading state to the development of ulcer, tissue-kallikrein was reduced and reduction of tissue-plasminogen activator was also observed.

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In disseminated intravascular coagulation, tissue thromboplastin (T. Tbp) plays a great role as one of the direct trigger substances.
Attempts were made to demonstrate the coagulation-fibrinolytic changes and ultrastructural changes of the lung after the injection of T. Tbp.
T. Tbp was injected into pigs and blood samples were taken 10sec, 1min and 5min after the start of the injection. Ten sec after the injection, APTT and TEG-r shortened and fibrinopeptide A increased remarkably. One min after the injection, whole plasmin activity increased and antiplasmin decreased. Thus, it was suggested that fibrin deposition in pulmonary capillaries were induced by the action of thrombin on fibrinogen.
Then, the lungs of rabbits were examined by electron microscope. In the lung of suddenly died rabbit after the injection, capillaries were filled with plate-lets and fibrins. Endothelial cells were damaged and interstitial tissues were edematous. Twenty four hrs after the injection, endothelial cells were damaged, but no fibrin fibers were seen. In the lungs of rabbits injected with T. Tbp once a week for weeks, endothelial cells were strongly damaged and thrombi composed of platelets and fibrins were observed. Interstitial tissues were edematous and lamellar inclusion bodies of type B alveolar epithelial cells were lost.
As explained above, the injection of T. Tbp brought remarkable injuries in pulmonary capillaries and surrounding tissues, causing pulmonary microthromboembolism.

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Both tissue thromboplastin and pulmonary surfactant show electron-microscopically lamellar structure and belong chemically to lipoprotein. Based on the assumption that the thromboplastic activity of the amniotic fluid is mainly due to pulmonary surfactant in the amniotic fluid, this study was done and the following results were obtained: (1) Pulmonary surfactant isolated from the amniotic fluid shortened the activity of plasma recalcification time (PRCT) with dose response. (2) The thromboplastic activity of the amniotic fluid increased with advancing gestational ages. (3) Shortening ratio of PRCT calculated from the following formula, (control PRCT-test PRCT)/control PRCT, was more than 32% when surfactant phospholipid was more than 1mg/dl of amniotic fluid which indicates sufficient maturation of the fetal lung. (4) There was a close correlationship between shake test, a simple rapid screening test for assesment of fetal lung maturity, and shortening ratio of PRCT. (5) From the neutralization effect of heparin on thromboplastic activity of amniotic fluid, 5 units of heparin were calculated to be sufficient to neutralize the thromboplastic activity of 1ml of amniotic fluid. This calculation will be of help in heparin therapy of amniotic fluid embolism.

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Peripheral and bone marrow leukocytes from 24 leukemic patients (including 5 patients with DIC) were analysed for coagulant and fibrinolytic activity. Suspension of leukocytes were prepared by dextran method. Washed preparations were disrupted by freezing and thawing three times and homogenized. Lysates of these leukocytes were added into prothrombin time system instead of lyoplastin (rabbit lung tissue thromboplastin). For comparison, leukocytes from 10 normal donnors were tested. Antithrombin III, fibrinogen, prothrombin and FDP were simultaneously measured and these change in clinical courses were compaired with leukocytic thromboplastin like activity.
Lysate of these leukocytes from peripheral and bone marrow in cases of leukemia with DIC showed high coagulant activity in prothrombin time system. FDP increased and antithrombin III, prothrombin and fibrinogen decreased. However lysate of leukemia without DIC showed normal or lower leukocytic thromboplastin like activity in prothrombin time system and FDP, antithrombin III, prothrombin and fibrinogen were not so changed. The coagulant activity of the lysate of AMMoL was intermediate between DIC and non DIC. In clinical courses of leukemic therapy, leukocytic thromboplastin like activity was relatively related to number of leukemic cells. The cases with high leukocytic thromboplastin like activity before chemotherapy have an anxierty to induce DIC.
We think that leukocytic thromboplastin like activity is related to DIC in leukemia.

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Measurements of Factor X clotting activity by using tissue thromboplastin (T. T.) and Russell's viper venom (R. V. V.) showed the correlated result for normal rabbit plasma and human plasmas from healthy persons and patients with liver disease. However the clotting activity of Factor X in plasmas from the rabbits and patients given warfarin become variable according to the assay methods, the activity assayed by T. T. was decreased, whereas it was not so by using R. V. V.
Plasma samples are tested in a series of dilutions (D) and the coagulation time (t) measured with assay system using T. T., R. V. V. and hepaplastin test (H. T.).
When t is plotted against D, a straight line was obtained. Extrapolation to infinite substrate concentration gives the minima coagulation time (t_min). Normal or liver disease plasma display the same value for t_min, when using any assay systems. Plasma from persons or rabbits given warfarin display the same value for t_min when using R. V. V. or H. T., but not by T. T.
These results show that clotting activity of Factor X assayed by T. T. is disturbed with competitive inhibitors probably Protein Induced by Vitamin K Absence or Antagonists (PIVKA) but assayed by R. V. V. or H. T. is not inhibited, as suggested by HEMKER.
The value of amidolytic activity of Factor X is higher than that of its clotting activity assayed by R. V. V. for plasma from the persons or rabbits given warfarin.
It is likely that there might be some substances which have no clotting activity but amidolytic activity associated with Factor X.
Further studies might be necessary to clarify the relationship between the clotting activity and the amidolytic activity of Factor X.

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Canine prothrombin was purified through following procedures: Combination of BaSO₄ adsorption, elution of adsorbed prothrombin in 0.25M sodium citrate, concentration of the eluate by (NH₄)SO₄ fractionation at 80% saturation and DEAE-cellulose chromatography.
The prothrombin preparation thus obtained was proved to be pure by immunoelectrophoresis but some trace impurities were visible by SDS polyacrylamide gel electrophoresis (SDS PAGE).
The molecular weight was 81, 300±850 (SD) on SDS PAGE, the extinction coefficient was 13.8±0.4, and the specific activity was 1, 200±50 NIH units per mg prothrombin.
Then, the activation behavior of prothrombin by activated Factor X, in the presence of Factor V, phospholipids and calcium was determined by SDS PAGE. The molecular weights for P₂, Fa, P₃ and Fb were 60, 000, 28, 540, 40, 400 and 21, 240 respectively.
Next, thrombin was separated by using QAE-Sephadex A50 chromatography. The molecular weight of thrombin averaged 38, 111±500, the extinction coefficient was 16.0±0.2 and the specific activity was 2, 000±65 NIH units per mg thrombin. One peculiar finding with the canine material was that P₂ appeared to be double band in SDS PAGE. Further studies are required to elucidate its significance.

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Two-dimensional immunoelectrophoresis of prothrombin was carried out on plasma samples of 6 month old patient with secondary vitamin K deficiency and thirty seven young infants aged one month.
In the patient with secondary vitamin K deficiency, abnormal prothrombin, which showed faster mobility in the presence of calcium ions, already appeared at 72 hours before onset of G. I. bleeding and coagulation disorder due to vitamin K deficiency, although normal prothrombin level was within the normal limits (Fig. 1, 2).
At 48 hours before onset, abnormal prothrombin increased to the same level as normal prothrombin which had decreased (Fig. 2).
Thereafter, coagulation disorder due to vitamin K deficiency appeared, because of lower level of normal prothrombin.
Thus, we could detect the mild vitamin K deficient state by two-dimensional immunoelectrophoresis of prothrombin, before the actual bleeding episode or prolongation of prothrombin time, Thrombotest and Hepaplastintest took place.
We found that about one-third of young infants aged one month had various level of abnormal prothrombin regardless of variety of feeding method, although normal prothrombin level was within the normal limits (Fig. 3). Based on these data, we concluded that about one-third of young infants aged one month were in mild vitamin K deficient state.
Those mild vitamin K deficient state could not be detected by the ratio of procoagulant activity to antigen of prothrombin, Thrombotest and Hepaplastintest (Fig. 4, 5).

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We have already reported that tranexamic acid (t-AMCHA) has influence on variation of tubidity accompaning fibrin formation. The addition of 0.5% t-AMCHA to fibrinogen increased the tubidity of fibrin. That was decreased in the case of 10% t-AMCHA. These phenomena occured even if factor XIII free fibrinogen was used.
In this study, the incorporation of t-AMCHA into fibrin and the participation t-AMCHA in fibrin polymerization were examined by TLC, SDS disc-electrophoresis (SDS PAGE) and monochloroacetic acid lysis time test, using factor XIII free fibrinogen (Daiichi).
The results are as follows;
1) By TLC, the incorporation of t-AMCHA in fibrin was suggested.
2) By SDS PAGE, both 0.5% and 10% t-AMCHA added fibrinogen showed only α, β, γ-chains, no γ-dimer and α-polymer.
3) By monochloroacetic acid lysis time test, lysis time was 17.7sec for 0.5% t-AMCHA and 90.0sec for 10%.
Standard lysis time was 8.5sec.
These results suggest that t-AMCHA participates in fibrin polymerization.

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Characteristics of active prothrombin complex (Factor VIII inhibitor by-passing activity: FEIBA) was investigated from viewpoint of blood coagulation.
Results are as follows:
(1) FEIBA (100units/ml) showed procoagulant activity of factors II (prothrombin), VII, IX and X. The existence of factor Xa and thrombin was also observed in its preparation. Prothrombin in its preparation was activated to thrombin with thromboplastin-calcium solution and in 2.5 molar glycine solution.
(2) FEIBA showed correcting power of coagulation disorders of factor VIII inhibitor plasma.
(3) Infusion of 50 units of FEIBA made rabbits died from occurrence of DIC.

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A 71 years old female patient with sigmoid cancer complained of rapidly growing meteorism, abdominal pain and tarry stool and was proved to have acquired factor V inhibitor producibility together with severe anemia, depression of platelet aggregability and marked prolongation of Stypven time. This inhibitor was revealed to belong to IgG which showed its inhibitor activity on factor V in normal plasma rather slowly and diminished by a factor VIII inhibitor bypassing activity preparation from blood: FEIBA in vivo as well as in vitro.
She was administered FEIBA intravenously and her hemorrhage cured remarkably for some while, but she expired soon when melena and the other hemorrhagic symptoms reoccurred to put her in hemorrhagic shock state.
The action of FEIBA was prominently strengthened by the addition of phospholipid with tissue thromboplastin component and calcium ion; so thus prudent control of the phospholipid level in plasma at the clinical application of this preparation was evaluated to bring good clinical effect and to avoid serious side effects such as hemorrhage, shock, defibrination syndrome, etc.

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Intimal injury was induced by inserting polyethylen tubing into the aorta of New Zealand white rabbit. The injury was restricted to the intima without interruption of the intimal elastic lamina. Platelet adhesion and aggregation were investigated by scanning or transmission electron microscopy. Microthrombus formation mainly composed of platelets was scatteredly observed at the injured areas.
More deep injury was induced by prickling a needle from the adventitia of the thoracic or abdominal aorta after thoracotomy or laparotomy, and scratching the opposite intima to the prickling site. The internal elastic lamina was interrupted and the deeper injury reaching the media of the aorta was induced. This injured area revealed prominent fibrin formation accompanied by adhesion of platelets on the fibrin network individually or in clusters.
Fibrin or fibrinogen coating on the surface of the platelet thrombi was demonstrated by immunohistochemical technique. These fibrin or fibrinogen was thought to play a significant role for the fixation and growth of the mural thrombi.
Therefore, we should take account of the treatment which promotes the fibrinolysis as well as the treatment which prevents the platelet aggregation when we treat the thrombosis.

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Changes of lymph flow, and levels of its fibrinolytic factors were studied with some female rabbits which suffered from scalding injury in their paw. Effects of previous administration of tranexamic acid or hydrocortisone were also evaluated. The results obtained are as follows.
1. Lymph flow was markedly increased after scalding, but the previous administration of tranexamic acid or hydrocortisone, particularly the latter, prevented the increase.
2. SK-activated plasminogen in local lymph elevated transiently at the earlierstage of scalding injury, and then gradually decreased, but showed a re-elevation tendency at the later stage. This change was similar to that of prostaglandin (PGE₁) after scalding as reported by us (to be published). By the administration of tranexamic acid, SK-activated plasminogen was more remarkably decreased in middle stage.
3. The levels of plasmin inhibitor were rapidly increased soon after the scalding. By the previous administration of tranexamic acid, however, the levels showed comparatively gradual increase but they continued to keep high activity for a long time.
These results suggest that changes of flow and of fibrinolytic factors in local lymph after scalding injury, are closely linked to some of the mediators involved in the inflammation. They also help reassure the antifibrinolytic effect of tranexamic acid.

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Burn injuries cause dynamic alterations of the coagulative and fibrinolytic activities of the blood. But there has been little research in this field.
In our research of burned patients who sustained burns over 30% of the total body surface area, platelet counts, PT, PTT, fibrinogen, ELT, fibrinolytic activities on various fibrin plates, FDP, α₁-antitrypsin, α₂-macroglobulin and antithrombin III were checked.
The results show that intravascular coagulation occurs soon after burn injury and it takes almost one month, including the passage through the hypercoagulative stage to normalize. So we devided the postburn period into 4 stages from the hematological standpoint.
Ist stage; within 48 hours. IInd stage; 3rd-7th day.
IIIrd stage; 8th-30th day. IVth stage; after 30th day.
We used heparin of 10, 000-20, 000U in dose on burned patients clinically to prevent the DIC in the Ist stage. From our experience, heparin therapy to burned patients is very effective and safe. But in severly burned patients, administration of FOY is recommended.

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A 32 years old female with Hermansky-Pudlak syndrome and brain tumor was reported. She was an albino with brown hair, white skin and constant horizontal nystagmus. She had suffered from nasal bleeding since childhood and had postoperative bleeding at the age of 31. There were no relatives with bleeding tendency or albinism but her parents were first cousins.
Bleeding time by Ivy method was more than 30 minutes. Platelet retention in glass-beads column was normal. Platelet aggregation studies showed the absence of the second stage of aggregation induced by adenosine diphosphate, defective aggregation by collagen and epinephrine, and normal by ristocetin. Adenosine diphosphate release of patient's platelets induced by kaolin was diminished. Bone marrow smear revealed abnormal pigmented histiocytes.
She was treated with cryoprecipitate (800 units of AHF). Within 30 minutes of infusion, bleeding time shortened to 12.5 minutes but this effect had disappeared after 2 hours. The abnormal platelet aggregation tests remained unchanged during this infusion study, Factor VIII clotting activity, factor VIII related antigen and von Willebrand factor were not correlated with bleeding time.

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Three-year-old boy with severe factor IX dificiency had developed frequent vomiting seven days after minor head injury. CT scan and CAG revealed a large intracranial hematoma in the left temporal lobe. Evacuation of the hematoma was performed. The factor IX concentrate was given to keep the plasma levels of factor IX at over 100% during operation and for 24 hours postoperatively, at 60% for following 6 days, and discontinued on 27 days after operation. Postoperative course was uneventful.
Our retrospective study on total Hemophiliacs (number 153; Hemophilia A 132, B 21), we experienced 23 cases of intracranial bleeding in 13 patient (Hemophilia A 11, B 2). 13 of 23 cases of intracranial bleeding had a history of recent head trauma, two thirds of which had a long symptom-free interval (mean 4.8 days).
Minor head trauma happens to cause rupture of intracranial small vessels, and continuous bleeding does not cease spontaneously in these patients to make a large intracranial hematoma during latent period. The prompt replacement therapy after head trauma even in the absence of symtom is the most important procedure for preventing intracranial bleeding.

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1. Bone marrow cells of mouse given endotoxin (LPS-cells) accerelated the plasma recalcification time (RCT) and partial thromboplastin time (PTT) of mouse or rabbit plasma. PTT was more sensitive than RCT. 2. A significant procoagulant activity of LPS-cells was demonstrated as early as 4hr and the peak was found 8-18hr after endotoxin. This peak was coincided with those of the cytotoxicity and the decrease of nucleated cell counts in marrow. 3. Either the procoagulant activity of LPS-cells of the mouse or the activity of mouse brain thromboplastin (MBT) was more marked in mouse plasma than in rabbit plasma. There would be a species specificity between the two animals, as to coagulation. 4. The two regression lines of the PTT on either the number of LPS-cells or the dose of MBT were nearly parallel. 5. Dose response regression of cytotoxicity or generation of procoagulant activity on concentration of endotoxin injected was significant. Cytotoxicity and procoagulant activity of LPS-cells were demonstrated even by injection of as little as 5μg endotoxin. 6. There existed a definite correlation between the cytotoxicity and procoagulant activity of LPS-cells (n=20, r=-0.8452, p<0.001).
These findings suggested that procoagulant activity of LPS-cells is shown to be tissue thromboplastin in nature, and the migrated-cells in circulation play an important role in blood coagulation of endotoxicosis.

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The existence of inconsistency between clinical and pathological diagnosis of DIC has been pointed out by many authors. Present study was undertaken to clarify this difference and to establish a reliable hemostatic criteria for diagnosis of DIC. A retrospective analysis was done by the investigation of twenty-six autopsies out of 64 cases with DIC which had been clinically diagnosed in our laboratory of Kyushu University School of Medicine from November, 1972 to May, 1979. There were seen malignancies in 22 (85%) and severe infections in 19 (73%), and combination of both diseases in 13 cases (50%) as the underlying diseases of this series. These cases were divided into two groups according to the presence of multiple fibrin thrombi as the pathological evidence of DIC. Microthrombi were detected in 14 cases (Group A), but not in 12 cases (Group B).
Decrease of platelet counts, plasminogen and antithrombin-III levels, prolongation of prothrombin time and serial thrombin time, increase of fibrin degradation products (FDP) levels and positive ethanol gelation test were seen in both groups. In these parameters, a significant difference in both groups was seen in only FDP levels, i. e. mean values were 88 and 36μg/ml respectively. In addition, microthrombi were not often detected when a hemostatic criteria of depleted platelet counts less than 100, 000/mm³, increased FDP levels more than 40μg/ml and positive ethanol gelation test are satisfied. Therefore, such a strict criteria shold be recommended in order to make a clinical diagnosis coincidental to pathological evidence of DIC.

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Complement level was studied in 13 cases of disseminated intravascular coagulation (DIC) patients associated 4 with severe bacterial infection, 1 with chronic myelogenous leukemia (CML) and 8 with liver diseases. Immediately after the onset of DIC, significant falls of CH50, C4 and C3 levels were observed in all cases. By the treatment of DIC mainly with heparin, those cases who failed to be cured maintained their low serum complement levels while those who recovered revealed gradual increase in serum complement.
Although it has not been fully studied whether complement plays a role in the pathogenesis of DIC, the present studied seem to suggest that the investigation of complement, besides coagulation factors and fibrinogen/fibrin degradation product (FDP), might provide clinical value for the early detection of the onset of DIC and for estimating the prognosis of the patients.

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Heparin concentration was measured with the clotting assay method. The assay was shown by the clotting time that the clotting of the thrombin, fibrinogen and antithrombin III mixture was prevented by addition plasma with heparin activity. The normal plasma of heparin concentration has the activity of mean value of 0.02±0.16 units. After 5, 000 units of heparin in normal, the peace value appeared after 10 minutes and the value gradually fell to the previous after 90 minutes. And the combination of one shot of 5, 000 units and continuous infusion of 10, 000 units for 24 hours was shown at 10 times higher average concentration. This combination use of heparin was thought to get easily the adequate concentration in plasma. But the effect of heparin after the infusion was influenced with antithrombin III activity. In the case of low a activity of antithrombin III, particularly of its biological activity, for instance DIC, the AT III level did not return to normal range in spite of high dose of heparin and the effect of heparin did not appear.

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Monospecific antibody against human platelet tubulin was prepared. Human platelet tubulin was isolated by three successive cycles of polymerization using 5×10¹¹ platelets obtained from healthy volunteers by Haemonetics Model 30 blood processor. Densitometric quantification of purified tubulin subjected to SDS-polyacrylamide gel electrophoresis revealed purity of 96%. Purified tubulin 400μg was given to rabbits with Freunds' adjuvant once a week four weeks. The rabbit anti-tubulin serum gave a single precipitin line on double immunodiffusion against platelet tubulin and the high speed supernatant of a platelet sonicate (platelet extract). Anti-tubulin F(ab′)₂ fragment was prepared from anti-tubulin rabbit serum. Platelets preincubated with anti-tubulin F(ab′)₂ showed decreased aggregation by collagen, but not by ADP or epinephrine.
These results may suggest that tubulin, possibly membrane-associated one, plays important roles in the mechanism of platelet aggregation induced by collagen.

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The changes in platelet aggregation and serum levels of lipid fractions were observed in 33 patients with arteriosclerosis prior to and after the 2 monthadministration of dl-α-tocopheryl nicotinate (600mg/day) or placebo.
Platelet aggregation by 2.5μM ADP (final concentration), 2μM epinephrine, 3μg/ml collagen and 0.5mM arachidonic acid decreased significantly, compared to that prior to the administration (p<0.02, p<0.05, p<0.05, and p<0.001, respectively). The level of hematocrit and platelet counts did not change during this period. The serum level of vitamin E increased from 1.23±0.46 (M±SD)mg/dl to 2.18±0.55mg/dl after the administration of dl-α-tocopheryl nicotinate. By the administration of this drug, the serum level of total cholesterol in patients with its initial level higher than 220mg/dl was decreased from 242.2±19.0mg/dl to 215.9±27.6mg/dl (p<0.01), and that of triglyceride was decreased from 214.0±68.1mg/dl to 170.7±53.8mg/dl (p<0.05) in patients with its initial level higher than 150mg/dl, while there was no significant change in the serum level of phospholipid.
This drug could be expected to be useful clinically for the prevention of arteriosclerosis due to a reduction in platelet aggregation and an improvement in lipid metabolism.

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Glu-plasminogen (Glu-plg) was better activated by urokinase (UK) in the presence of fibrin or tranexamic acid. Lys-plg was not influenced by their presence, and acid treated plg (A-plg) was less activated by UK in their presence. Inhibitory effects of α₂ plasmin inhibitor (α₂PI) on plasmin were counteracted by fibrin, tranexamic acid and α₂ macroglobulin (α₂M). Since fibrin, tranexamic acid, and α₂PI bind with lysine binding sites of plasminogen, there are competitions for these sites among α₂PI, fibrin, and tranexamic acid.

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It is well known that FDP appears in urine of patients with glomerulonephritis. To clarify the problem of whether urinary FDP arises from the lysis of the glomerular fibrin deposits or by filtration of fibrinogen and FDP from the plasma, we have studied the presence of D-dimer in serum and urine from patients with various renal disease.
FDP from plasma and urine was isolated by chromatography using small columns of activated Sepharose 4-B coupled with antifibrinogen Serum, and analysed by SDS-polyacrylamide gel electrophoresis.
1. D-dimers in urine were found in 13 of 26 cases and those in plasma in 5 of 15 cases.
2. Cases with positive urinary D-dimer had higher levels of urinary FDP, proteinuria, micro hematuria and severe renal dysfunction in comparison with cases with negative urinary D-dimer.
3. Ratio of D-dimer to monomer in urinary FDP was about 10 times more than in serum FDP. On the basis of this data, it may be suggested that there is intraglomerular fibrinolysis in cases with positive urinary D-dimer.

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The role of intravascular coagulation in the progression of immune complex induced nephritis (BSA nephritis) was examined in the albino rabbits, and the effects of urokinase and ancrod were tested.
Non-treated group revealed hyperfibrinogenemia, high level of serum FDP and low plasminogen activator activity accompanying endocapillary proliferation after BSA treatment.
By daily infusion of urokinase (3, 000U/day), the activity of plasminogen activator was conserved and the glomeruli showed more mild lesions than that of non-treated group.
By ancrod infusion (2U/kg/day; iv), the degree of endocapillary proliferation decreased accompanying hypofibrinogenemia.
These results suggest that intravascular coagulation plays an important role of BSA nephritis.

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Larger amounts of FDP were found in the ascitic fluid associated with peritonitis carcinomatosa than in the non-malignant fluid. By Todd's fibrin slide method, the dissolution of fibrin was observed around free cancer cells in the ascites, but not around the granulocytes. Therefore, it was suggested that the origin of FDP in malignant ascitic fluid was the plasminogen activator dependent on the cancer cells.
By Lysine-Sepharose affinity chromatography, plasminogen activator was partially purified 4, 500 folds from ascitic fluid with peritonitis carcinomatosa of breast cancer (FDP 1, 280μg/ml). Euglobulin fraction of the malignant ascites was applied to the column on Lysine-Sepharose 4B, and eluted with 0.5M NaCl, 1M NaCl and 0.1M acetic acid. The activator was observed in the fraction of 1M NaCl elution, and plasmin in the fraction of 0.1M acetic acid.
On Ouchterlony's method, a precipitin line was not observed between the activator and anti-urokinase rabbit serum.
This result indicates that this plasminogen activator might be different from urokinase.

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Several multimolecular forms have been reported in human native plasminogen. This paper deals with the assessing method for multiforms of plasminogen, and multiform patterns obtained in healthy adults, newborns and patients with renal disease.
The multiforms in individuals were assessed by disc gel electrophoresis with continuous buffered system containing EACA. As the mobility of each subform to myoglobin was highly reproducible, the mobility of each form of plasminogen from various origin could be compared. Purified plasminogen from most of persons was consisted of five major native plasminogens and a few minor ones. Furthermore, the mobility of each subform was identical in three groups. The results obtained suggested that the neonatal and adult plasminogen might be synthesized by the same gene.

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The present study was carried out to determine which proteases could induce the release of plasminogen activator from blood vessels of isolated dog hind leg perfused with artificial physiological solution. Proteases tested were thrombin, trypsin, chymotrypsin and F. Xa which perform limited proteolysis and papain, which proteolyzes less selectively. Thrombin released plasminogen activator dose-dependently at the infused concentrations between 2×10^-8 (M) and 6×10^-7 (M). Trypsin also released plasminogen activator as strongly as thrombin. On the other hand, chymotrypsin had little effect on plasminogen activator release even at 5×10^-6 (M). F. Xa had no activity to release plasminogen activator at the concentrations between 10^-7 (M) and 5×10^-7 (M). The weak effect of papain on plasminogen activator release was observed but considered not to be specific. These results indicate that plasminogen activator release from vessel wall can not be induced by every protease generally but by only particular proteases such as thrombin and trypsin specifically. This may imply the participation of certain Arg residue of the reactive site on the vessel wall in the release of plasminogen activator by proteases.

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The purpose of this investigation is to study the enzymatic properties of rat bone marrow extract which exhibits high fibrinolytic activity, and to observe whether or not the activity is influenced by the whole body irradiation.
The results were obtained as follows:
1) A high fibrin degradation activity was extracted with 2M KSCN from rat bone marrow, but failed with 0.15M NaCl or 0.88M sucrose.
2) The activity was clearly demonstrated on plasminogen-free fibrin plates, whereas it was not shown on plasminogen-rich fibrin plates. Assumingly, the activity differs with plasminogen activator.
3) The activity was strongly inhibited with STI as well as DFP, while it was bearly inhibited with TLCK, t-AMCHA and Aprotinin. On the bases of above results, the factor extracted from rat bone marrow by us appears to be identical or similar to the factor in the conventional rat spleen recently found by Okamoto and Nagamatsu.
4) The activity of rat bone marrow extract was remarkably decreased by γ-ray whole body irradiation, and the observed tendency was well correlated with the irradiation-induced bone marrow damage. The activity is, therefore, implicated to be a bone marrow cell origin.

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The localization of plasminogen activator in ovulation was investigated using the fibrin slide method. In regular cycle rats, plasminogen activator activity appeared in the external area of the follicle wall (stigma) from 12 hours before ovulation. No activity was noticed in the follicular cavity at this time. A peak of activity was seen at 2 hours before ovulation. After ovulation, the activity decreased markedly. The activity was completely inhibited on fibrin slides to which 10^-2M t-AMCHA had been added. No activity was apparent on plasminogen-free fibrin slides.
The plasminogen activator activities on the follicular wall and in the follicular fluid of swine were also measured using the fibrin plate method. The activity on the follicular wall increased from the diestrous to the estrous phase. No activity was apparent in the follicular fluid at any phase.

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A study was made on individual components of the fibrinolytic system and on the level of the degradation products of fibrinogen and fibrin (FDP) in children, mainly in pediatric patients. Plasminogen and whole inhibitor in the blood before and after the operation, were determined by an affinity chromatography and TNP method. FDP in the blood was determined by a latex agglutination test. The value of plasminogen was no difference between in healthy children and in adults and aged people, but the value of inhibitor was generally higher in healthy children than in adults and aged people.
Seventeen pediatric patients undergoing surgical procedures of various types were not induced significant change in plasminogen and inhibitor by surgical operation. From the viewpoint of a disease, the values of both plasminogen and inhibitor in patients with neuroblastoma were very low. An abnormal level of FDP was observed in patients with leukemia or hepatoblastoma.

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When plasmin was mixed with α₂M or α₂PI, plasmin was inhibited faster by α₂PI than by α₂M. When plasma was mixed with UK and fluorescamine-labeled casein (f-casein), hydrolysis of f-casein was observed 10min after incubation started, and increased significantly after 20min incubation. Crossed-immunoelectrophoresis indicates the presence of free α₂PI and α₂PI-plasmin complex at 10, and 30min incubation. These results indicate that plasmin, α₂PI and α₂PI-plasmin complex could coexist in the plasma in contrast to a purified system.

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Both activation and inhibition of proteolysis are considered to regulate tissue reactions in inflammation. Present studies investigate inhibitors against fibrinolytic processes using synthetic chromogenic substrates in the extract of granulomatous tissue in schistosomiasis. Hepatic extracts prepared from schistosomeinfected mice demonstrated rapid, time dependent, stoichiometric and irreversible inhibition of urokinase; extracts from noninfected mice, however, elicited negligible inhibition. A small amount of plasmin inhibition was shared by both noninfected and infected mice. Pattern of Sephadex G-200 gel-chromatography of an ammonium sulphate fraction illustrates two peaks exhibiting urokinase inhibition, distinguishing an antiplasmin peak. Apparent induction of urokinase inhibitors in the granulomatous tissue accounted for increase in the whole antifibrinolytic activity previously measured by lysis time assay of fibrin clots.
In frozen sections of the liver, fibrin was found to deposite on immature granulomas by use of immunofluorescence microscopy. However, deposited fibrin was cleared at the center of each lesions when glanulomatous inflammation fully developed.
We postulate, therefore, that the proteinase inhibitor in tissue limits activation of the local fibrinolysis, and subsequently may regulate the development of chronic granulomatous inflammation.

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Plasmin inhibitor was obtained from a marine red algae, Grateloupia livida (G. livida) and Grateloupia elliptica (G. elliptica), in purified by ammonium sulfate fraction, gel filtration on Sephadex G-100, followed by ion exchange chromatography on DEAF-Sephadex and affinity chromatography on Trypsinporous glass and/or Plasmin-porous glass column.
A specific protein —an inhibitor of human plasmin— was isolated from G. livida and G. elliptica in a homogenous from. The protein, isolated from red algae, did not inhibit papain and pepsin. The inhibitor are also effective toward trypsin. The inhibitors were almost completely inactivated by heating at 100°C for 10min, and the inhibitors seemed fairly resistant for temperature.
The inhibitor was obtained by affinity chromatography shows remarbable stability to high temperature (100°C for 10min). The inhibitor is also effective toward trypsin. It is, however, digested by paparin and pepsin.
Further investigation to clarify the physiological function of these serine proteinase inhibitors in marine algae now in progress.

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Fibrinolytic system in the arterial wall seems to play an important role in the development and progression of atherosclerosis and in the formation and resolution of thrombi. Vascular plasminogen activator has been reported to be produced and secreted by endothelial cells, but fibrinolytic inhibitor in the arterial wall has not been fully investigated. Therefore we studied the inhibitor of fibrinolysis in human arterial wall.
Results:
1. Inhibitor of fibrinolysis was found in the intima and media of human arteries, and also in the atherosclerotic lesion.
2. Acetone powder of the intima and media of human aorta was extracted with cold saline and partially purified by the procedures including ammonium sulfate fractionation, Concanavalin A-Sepharose affinity column, DEAE-Sepharose CL-6B column, and immunoadsorbent column chromatography.
3. The final preparation was shown to be immunologically different from known serum protease inhibitors, and suggested to be a glycoprotein originated from tissue.
4. The inhibitor suppressed the fibrinolysis induced by urokinase and partially purified tissue plasminogen activator, and hydrolytic activity of urokinase directly.
This inhibitor may play a regulatory role on fibrinolysis in arterial wall and may play a role in the development and progression of atherosclerosis.

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